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We have generated putative promoter tagged transgenic lines inArachis hypogaea cv JL-24 using cotyledonary node (CN) as an explant and a promoterless gus::nptII bifunctional fusion gene mediated byAgrobacterium transformation. MS medium fortified with 6-benzylaminopurine (BAP) at 4 mg/l in combination with 0.1 mg/l α-napthaleneacetic
acid (NAA) was the most effective out of the various BAP and NAA combinations tested in multiple shoot bud formation. Parameters
enhancing genetic transformation viz. seedling age,Agrobacterium genetic background and co-cultivation periods were studied by using the binary vector p35SGUSINT. Genetic transformation
with CN explants from 6-day-old seedlings co-cultivated withAgrobacterium GV2260 strain for 3 days resulted in high kanamycin resistant shoot induction percentage (45%); approximately 31% transformation
frequency was achieved with p35S GUSINT in Β-glucuronidase (GUS) assays. Among thein vivo GUS fusions studied with promoterless gus::nptII construct, GUS-positive sectors occupied 38% of the total transient GUS
percentage. We have generated over 141 putative T0 plants by using the promoterless construct and transferred them to the field. Among these, 82 plants survived well in the
green house and 5 plants corresponding to 3.54% showed stable integration of the fusion gene as evidenced by GUS, polymerase
chain reaction (PCR) and Southern blot analyses. Twenty-four plants were positive for GUS showing either tissue-specific expression
or blue spots in at least one plant part. The progeny of 15 T0 plants indicated Mendelian inheritance pattern of segregation for single-copy integration. The tissue-specific GUS expression
patterns were more or less similar in both T0 and corresponding T1 progeny plants. We present the differential patterns of GUS expression identified in the putative promoter-tagged transgenic
lines in the present communication. 相似文献
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Jumonji C (JmjC) lysine demethylases (KDMs) are Fe(II)-dependent hydroxylases that catalyze the oxidative demethylation of methyllysine residues in histones and nonhistone proteins. These enzymes play vital roles in regulating cellular processes such as gene expression, cell cycle progression, and stem cell self-renewal and differentiation. Despite their biological importance, recombinant forms of JmjC KDMs generally display low enzymatic activity and have remained challenging to isolate in a highly active form. Here we present a simple affinity purification scheme for Strep(II)-tagged JmjC KDMs that minimizes contamination by transition state metal ions, yielding highly active and pure enzyme. We also describe an optimized continuous fluorescent assay for KDMs that detects formaldehyde production during demethylation via a coupled reaction using formaldehyde dehydrogenase. Purification and kinetic analysis of the human KDMs JMJD2A and JMJD2D using these methods yielded activities substantially higher than those previously reported for these enzymes, which are comparable to that of the flavin-dependent KDM LSD1. In addition, we show that JMJD2A exhibited a lower catalytic efficiency toward a histone peptide bearing a chemically installed trimethyllysine analog compared with a bona fide trimethylated substrate. The methodology described here is broadly applicable to other JmjC KDMs, facilitating their biochemical characterization and high-throughput screening applications. 相似文献
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Crossovers (COs) between homologous chromosomes ensure their faithful segregation during meiosis. We identify C.?elegans COSA-1, a cyclin-related protein conserved in metazoa, as a key component required to convert meiotic double-strand breaks (DSBs) into COs. During late meiotic prophase, COSA-1 localizes to foci that correspond to the single CO site on each homolog pair and indicate sites of eventual concentration of other conserved CO proteins. Chromosomes gain and lose competence to load CO proteins during meiotic progression, with competence to load COSA-1 requiring prior licensing. Our data further suggest a self-reinforcing mechanism maintaining CO designation. Modeling of a nonlinear dose-response relationship between IR-induced DSBs and COSA-1 foci reveals efficient conversion of DSBs into COs when DSBs are limiting and a robust capacity to limit cytologically differentiated CO sites when DSBs are in excess. COSA-1 foci serve as a unique live cell readout for investigating CO formation and CO interference. 相似文献
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A high frequency in vitro shoot bud differentiation and multiple shoot production protocol from hypocotyl segments of 8 to 10-d-old seedlings of cotton
has been developed. Murashige and Skoog (MS) basal medium with Nitsch and Nitsch vitamins was found to be optimal in shoot
regeneration. A combination of 2 mg dm−3 thidiazuron and 0.05 mg dm−3 naphthaleneacetic acid was the most effective for shoot regeneration (76 %) and an average of 10.6 shoots per responding
explant. Combination of the cytokinins benzylaminopurine and kinetin induced better regeneration response than their individual
treatments. Supplementation of the culture medium with ethylene inhibitor silver nitrate and activated charcoal showed beneficial
effects. Optimal rooting was obtained on half-strength MS medium supplemented with 1 mg dm−3 indolebutyric acid and activated charcoal. Scanning electron micrographs of in vitro cultured explants revealed that shoot primordia were formed de novo. 相似文献
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Hiromi Sasaki Swathi V. Iyer Ken Sasaki Ossama W. Tawfik Tomoo Iwakuma 《Analytical biochemistry》2015
Osteosarcoma, the most common type of primary bone cancer, is the second highest cause of cancer-related death in pediatric patients. To understand the mechanisms behind osteosarcoma progression and to discover novel therapeutic strategies for this disease, a reliable and appropriate mouse model is essential. For this purpose, osteosarcoma cells need to be injected into the bone marrow. Previously, the intratibial and intrafemoral injection methods were reported; however, the major drawback of these methods is the potential leakage of tumor cells from the injection site during or after these procedures. To overcome this, we have established an improved method to minimize leakage in an orthotopic mouse model of osteosarcoma. By taking advantage of the anatomical benefits of the femur with less bowing and larger medullary cavity than those of the tibia, osteosarcoma cells are injected directly into the femoral cavity following reaming of its intramedullary space. To prevent potential leakage of tumor cells during and after the surgery, the injection site is sealed with bone wax. This method requires a minor surgery of approximately 15 min under anesthesia. Our established orthotopic osteosarcoma model could serve as a valuable and reliable tool for examining progression of various types of bone tumors. 相似文献
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Conserved insulin signaling in the regulation of oocyte growth,development, and maturation 下载免费PDF全文
Insulin signaling regulates various aspects of physiology, such as glucose homeostasis and aging, and is a key determinant of female reproduction in metazoans. That insulin signaling is crucial for female reproductive health is clear from clinical data linking hyperinsulinemic and hypoinsulinemic condition with certain types of ovarian dysfunction, such as altered steroidogenesis, polycystic ovary syndrome, and infertility. Thus, understanding the signaling mechanisms that underlie the control of insulin‐mediated ovarian development is important for the accurate diagnosis of and intervention for female infertility. Studies of invertebrate and vertebrate model systems have revealed the molecular determinants that transduce insulin signaling as well as which biological processes are regulated by the insulin‐signaling pathway. The molecular determinants of the insulin‐signaling pathway, from the insulin receptor to its downstream signaling components, are structurally and functionally conserved across evolution, from worms to mammals—yet, physiological differences in signaling still exist. Insulin signaling acts cooperatively with gonadotropins in mammals and lower vertebrates to mediate various aspects of ovarian development, mainly owing to evolution of the endocrine system in vertebrates. In contrast, insulin signaling in Drosophila and Caenorhabditis elegans directly regulates oocyte growth and maturation. In this review, we compare and contrast insulin‐mediated regulation of ovarian functions in mammals, lower vertebrates, C. elegans, and Drosophila, and highlight conserved signaling pathways and regulatory mechanisms in general while illustrating insulin's unique role in specific reproductive processes. 相似文献
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PprA is known to contribute to Deinococcus radiodurans'' remarkable capacity to survive a variety of genotoxic assaults. The molecular bases for PprA''s role(s) in the maintenance of the damaged D. radiodurans genome are incompletely understood, but PprA is thought to promote D. radiodurans''s capacity for DSB repair. PprA is found in a multiprotein DNA processing complex along with an ATP type DNA ligase, and the D. radiodurans toposiomerase IB (DraTopoIB) as well as other proteins. Here, we show that PprA is a key contributor to D. radiodurans resistance to nalidixic acid (Nal), an inhibitor of topoisomerase II. Growth of wild type D. radiodurans and a pprA mutant were similar in the absence of exogenous genotoxic insults; however, the pprA mutant exhibited marked growth delay and a higher frequency of anucleate cells following treatment with DNA-damaging agents. We show that PprA interacts with both DraTopoIB and the Gyrase A subunit (DraGyrA) in vivo and that purified PprA enhances DraTopoIB catalysed relaxation of supercoiled DNA. Thus, besides promoting DNA repair, our findings suggest that PprA also contributes to preserving the integrity of the D. radiodurans genome following DNA damage by interacting with DNA topoisomerases and by facilitating the actions of DraTopoIB. 相似文献
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Brian M. Sandroff Robert W. Motl Lara A. Pilutti Yvonne C. Learmonth Ipek Ensari Deirdre Dlugonski Rachel E. Klaren Swathi Balantrapu Barry J. Riskin 《PloS one》2014,9(4)